Although the Human Genome was sequenced to 99.7% completion in 2003, it has become clear that the sequence of DNA alone is not sufficient to explain human health and disease in its entirety. This has led to a new appreciation of the factors outside the DNA sequence itself that regulate cellular processes and outcomes. In eukaryotic cells, the DNA is packaged into arrays of nucleosomes, each of which contains ~146 base pairs of DNA wrapped around an octamer of the core histone proteins. For almost half a century, nucleosomes were thought to be nothing more than building blocks, serving only to package and organize eukaryotic DNA within the nucleus. It is now apparent that histone proteins as well as the associated regulatory machinery are required for all nuclear processes. Chromatin dynamics play a central role in regulating gene expression and are necessary to maintain normal cellular function. Dysregulation of epigenetic signals has been associated with numerous human malignancies. Chromatin-regulating factors are some of the most frequently affected proteins in cancer, and these proteins have emerged as promising therapeutic targets. Many epigenetic regulatory mechanisms, including acetylation of lysine 56 in histone H3 (H3K56Ac), are conserved in eukaryotes. H3K56Ac plays a critical role in replication-dependent histone deposition, nucleosome turnover, DNA repair and in promoting pluripotency of human embryonic stem cells. Alternations in H3K56Ac levels have negative consequences on transcription and genome stability, and elevated levels of H3K56Ac have been measured in several human cancers. Together, the results indicate a regulated balance between acetylation and deacetylation of H3K56 is required for normal cellular function. Recent work suggests H3K56Ac levels are important for regulating noncoding transcription and chromosomal folding in yeast. However, it is unclear whether the effects are replication-dependent or -independent, making it difficult to fully understand the mechanism. The goals of this research are to investigate the role of replication-independent H3K56 acetylation in regulating transcription and chromosomal folding as well as to determine whether aberrant transcription caused by hyperacetylation ultimately leads to DNA damage. State-of-the-art genome-wide analyses of the transcriptome and genome architecture, including native-elongating transcript sequencing and a recently developed chromatin capture technique, termed Micro-C, will be utilized to monitor transcription and chromatin folding at single nucleotide and nucleosome level resolution, respectively. By using novel genome-spanning methods to answer complex biological questions, the results will provide important molecular insight into the cellular functions of H3K56 acetylation as well as the consequences of hyperacetylation, which may have important implications in understanding human malignancies. Furthermore, the results will greatly enhance our knowledge of the interplay between histone PTMs, chromatin remodeling, and chromatin structure as well as their roles in the mechanisms governing transcriptional control.